Detection and identification of acid-fast bacilli of Mycobacterium species by conventional procedures requires growing the organisms from patient specimens and then testing the isolates for various phenotypic characteristics. This laboratory previously reported the development of a polymerase chain reaction (PCR) and nucleic acid sequencing assay, targeting SecA1 housekeeping gene, that can be used to identify most mycobacteria at the species level. Additionally, preliminary studies indicated this test could be used directly with patient samples to detect and identify mycobacteria in clinical specimens, thus avoiding the lengthy delays involved with the traditional culture and identification methods. [unreadable] [unreadable] A total of 76 respiratory specimens (8 bronchial alveolar lavages, 68 sputa) collected and frozen at the NIH Mycobacteriology Laboratory were tested by our PCR SecA1 assay, with the results compared with acid-fast smears and cultures. A total of 69 of 76 specimens were positive for mycobacteria, and the SecA1 assay had a test sensitivity of 87% and specificity of 100%. The sensitivity of the assay was 100% for specimens with many acid-fast baciili seen by microscopy and 72% for the smear-negative, culture-positive specimens. Recently, a second study was initiated with specimens collected from patients in Uganda who presented to a Tuberculosis Clinic. Specimens were collected from approximately 140 patients and then shipped frozen to the United States. The NIH Mycobacteriology Lab processed all specimens by smear and culture, and we performed PCR analysis on the specimens. Approximately 65% of the patients had positive smears and cultures for mycobacteria. The initial analysis of the PCR data revealed a test sensitivity of 91% and specificityof 84%. Additional analysis is underway to determine if the discrepant results are related to prior antimycobacterial therapy in this patient population.